The long term goal of these studies is to unravel the molecular mechanism of action of the visual pigment rhodopsin. This work will lead to the design of specific, active-site directed, and/or mechanism-based reagents aimed at treatment of diseases such as retinitis pigmentosa and congenital stationary night blindness. There are two specific aims in this proposal: (i) to identify and elucidate the role of essential amino acid residues in rhodopsin, and (ii) to use this information to design active-site directed reagents that target mutant rhodopsins. The experimental approach will be to use site-directed mutagenesis combined with extensive in vitro functional analysis and chemical modification of the altered rhodopsins. Mutagenesis will be used to identify essential amino acids in the activation and inactivation of rhodopsin and opsin. Crosslinking studies will be used to determine how close together are essential residues in the active site. Transducin activity assays will be performed to determine how closely the activated mutants resemble photoactivated rhodopsin. Finally, the increased understanding of mechanism in the wild-type and mutant rhodopsins will be used to design active-site directed reagents based on retinal analogs that specifically target mutant rhodopsins found in patients with retinitis pigmentosa and congenital stationary night blindness.
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